우리 감사 Sean B. Carroll와 토마스 베르너 비행 주식, 장비에 대 한 나 오 유키 퓨즈, 촬영, 멘토링에 대 한 Kiyokazu 아가타와 엘리자베스 나카지마 영어 편집을 위한 그의 지원에 대 한 병 석 진 제공. 이 작품은 KAKENHI에 의해 지원 되었다 17 K 19427와 다케다 과학 재단.

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저자는 전혀 충돌의 관심이 없다.

설명 여기 번데기 단계의 정의 대 한 프로토콜 번데기 단계의 기간 및 디 guttifera에서 날개에 검은 반점의 강렬의 측정 측정 자세한 관찰에 대 한 puparium를 제거 합니다. 이러한 프로토콜 많은 초파리 에 대 한 적용할 수 있으며 비행 종, 특히 종 날개 색소와 관련.
깊이 있는 관찰과 자세한 개발 이벤트의 설명 것 하위 단계의 추가 사용. 많은 경우에, 요구 해 부 나는 번데기의 단면 발달 이벤트 단계 정의에 적합 하지 않습니다, 그리고 개별 어렵다 하나 준비에 대 한 번데기를 죽 일 하 여 추가의 사용 때문에. 새로운 초파리 의 사용에 대 한 첫 번째 단계로 발달 이벤트는 puparium 외부에서 구별할 수를 고용 해야 하나. 연구의 목적에 따라 하나 수 있습니다 다음 더 세분화 단계 특정 organogenesis 또는 다른 발달 이벤트에 따라.
여러 단계의 interspecific 비교에 대 한 잠재적인 어려움 (heterochrony27) 종 중 개발 이벤트 순서의 반전 이다. 예를 들어 D. melanogaster에서 말 관 된다 녹색 하 고 디 guttifera19의 일부 pupae에이 순서를 거꾸로 수 있습니다 반면 다음 노란색 몸 표시 됩니다 키를 누릅니다. 이러한 경우에 동종 단계 사이 엄격한 비교가 어렵습니다. 관심의 현상에 따라 하나를 다시 정의 하거나 발달 이벤트에 따라 특정 단계를 세분화 해야 합니다. 예를 들어 우리 수 대략 pupae P5-6의 선택한 번데기 날개 날개 형태를 사용 하 여 개발 타이밍14의 지표로 서 진 식의 interspecific 비교.
일반적으로, 그것은 puparium를 제거 하려면 10-30 분 걸립니다. 하나는 짧은 단계를 관찰 하 고 싶다면 pupae puparium 제거 하는 동안 전달 하는 시간을 고려해 준비 되어야 한다. 예를 들어 P12(i) D. guttifera, 유일한 1.1 h 기간에이의 관찰 하 고 싶어 하는 경우 P11에 번데기를 준비 하 고 좋은 결과 줄 것 이다.
우리의 프로토콜에서 moistened 휴지 번데기 이미지의 배경에 사용 됩니다. 관찰과 구조는 그림에 표시 하 고 싶어의 단계에 따라 하나는 흰색 또는 검은색 휴지를 사용할 수 있습니다. P4(ii) 전환 P3에 대 한 번데기에 거품의 위치는 중요 한 단계를 구별 하 고 검은 휴지 거품의 위치를 관찰 하는 데 도움이. P5 후 단계에 대 한 흰색 휴지 때문에 낫다 노란 몸, 눈 색깔, bristles, 그리고 색을 관찰 하는 데 도움이.
베인 브리지 및 Bownes17 외관의 그들의 주파수에서 번데기 단계 기간 추정. 그들의 준비 테이블은 D. melanogaster18하나 가장 널리 사용된. 그들의 방법에 대 한 그들은 5 성인 여성 및 5 명의 성인 남성을 포함 하는 4 개의 식품 병을 준비, 어둠 속에서 그들을 유지 하 고 pupae 무작위로 찍은 한 병에서 달걀 누워의 발병 후 11, 12, 13, 그리고 14 일에. 그들은 특정 단계에서 pupae의 수를 계산 하 고 평균을 계산. 번데기 단계 각의 길이 총 기간의 번데기 기간 데이터 및 이러한 주파수 데이터에 따라 추정 수 있습니다. 이 방법으로 한 문제는 그것은 사용할 수 없습니다 개발 타이밍 pupae 중 동기화 할 하는 경향이 경우 번데기 단계의 정확한 기간을 추정 하는.
사실, 우리 D. guttifera에서 베인 브리지 및 Bownes의 방법을 시도 하 고 우리가 번데기 들 동기화 때문에 편견된 데이터를 얻은. 우리가이 현상의 원인을 식별 하지 수 있지만 몇 가지 가능성은 1) 번데기 그들의 젊은 무대에서 circadian 리듬을 유지 하 고는 그리고/또한 2) 그들은 관찰에서 발생 한 빛에 노출에 반응. 따라서, 우리는 직접 관찰에 의해 실제 단계 기간을 측정 하기로 결정 했습니다. Circadian 리듬으로 인 한 편견을 최소화 됩니다.
여기 설명 하는 방법을 세 자리 지역 마약에서 컨트롤 영역의 빼서 반점 그들의 주변 제어 영역 (ΔOD)에 비해 멜 라 닌의 추가 누적 계량 하는 방법이입니다. 이 메서드는 Feulgen 얼룩 및 이미지 분석 (densitometry28,29) 하 여 핵 DNA 콘텐츠를 측정 하는 방법에 의해 영감을 했다. 날개 염색에이 방법을 적용 하기 위한 잠재적인 문제 중 하나로 서, ΔOD는 번데기는 아주 젊은 있으며 거의 착 색 하는 경우에 특히 음수 값을 수 있습니다. 후기, 컨트롤 영역에서 몇 가지 착 색 될 수 있습니다. 자리 지역 자체의 간단한 OD의 사용은 연구의 목적에 따라 ΔOD 대신 적절 한 수 있습니다. D. guttifera 경우 ΔOD 대신 간단한 OD의 사용 데이터의 추세 또는 연구의 결론 변화 하지 않았다.

초파리 의 형태학 특성 중 일부는 종1,2,3,,45중 다양. 우리는 질문에 접근할 수 있다 어떻게 형태학 상 다양성의이 형태학의 세대의 메커니즘을 비교 하 여 발생. 이러한 형태학의 예로 애벌레 trichomes, 성인 섹스 빗, 외부 생식 기 기구, 복 부 착 색, 및 날개 염색6,7,,89, 10 , 11 , 12 , 13 , 14 , 15. 성인 간의 형태학 적 차이 연구, 관찰 및 번데기 단계 분석, 때문에 중요 하다 성인 특성의 운명을 후반 애벌레 단계에서 결정 되 고 후속 morphogenesis 번데기 기간 동안 진행.
초파리 melanogaster의 발달 생물학 연구에서 "시간 APF" (번데기 형성 후 시간)16번데기 단계 표시 하는 일반적인 방법입니다. 이 시스템 번데기 형성 후 절대 시간을 고용 하 고 일상적인 실험에 대 한 매우 편리 하다. 그러나, 발달 속도 pupae, 사이 다를 수 있습니다 약간의 유전적, 후 또는 microenvironmental 차이 의해 영향을 받을 수 있고 따라서 같은 데 번데기 형성 후 절대 시간을 보장 하지 않습니다 pupae 동시는 발달 단계입니다. 대부분의 경우, 형태학 상 특징에 의해 정의 하는 단계는 여러 개인 비교를. 특히, 종 사이 비교에 해당 (동종) 단계 중 비교 정확한 준비 필요합니다.
베인 브리지 및 Bownes17 인식 20 번데기 단계 (P15(ii)) P1 초파리 melanogaster pupae의 형태학 적 특징에 따라. 이 발판은 형태학 상 발달 준비18의 가장 널리 사용 되는 시스템 이다. 이전 연구에서 우리는 날개 염색 연구19에 대 한 기초 확립 초파리 guttifera 의 번데기 준비 수행. D. guttifera 그것의 날개에 검은 물방울 패턴 있으며 날개 색소 형성20모델 종 중 하나입니다. 우리가 형태학 기준에 설명 된 간주 브릿지 Bownes' 연구17, 우리는 직접 직렬 관찰19, 베인 브리지 및 Bownes의 추정 단계 기간을 사용 하는 대신 단계 기간을 측정 주파수를 관찰 했다. 여기 우리가 번데기 준비의 방법 및 측정의 Fukutomi 외19에 사용 되는 초파리 의 번데기 단계의 기간을 설명 합니다.
날개 착 색의 개발 메커니즘 연구, 번데기 또는 성인 단계에서 색소 발생 시 알아야 합니다. Fukutomi 외. 19 날개 이미지의 이미지 분석에 의해 번데기 및 성인 단계 동안 착 색의 광학 밀도 (ODs)을 측정할. 초파리 날개의 착 색 검은 멜 라 닌21의 축적으로 인 한 것으로 생각 된다. ODs의 정량화, 그레이 스케일 이미지와 ImageJ 소프트웨어 (https://imagej.nih.gov/ij/)22 사용 되었다. 인식 하 고 계량 자리 전용 착 색 (ΔOD), 우리는 세 자리 세 자리 내부에서 외부 감. 확인 하려면이 방법을 재현 하 고 객관적인, OD 측정의 랜드마크로 서 날개 정 맥을 사용 하 여 결정 한다. 이 문서에서는, 우리가 자세히 설명 초파리 guttifera착 색 날개의 정량화의이 방법.

<table>
	<tbody>
		<tr>
			<td>Drosophila guttifera</td>
			<td>The Drosophila Species Stock Center at the U.C. San Diego</td>
			<td>15130-1971.10</td>
			<td>Drosophila guttifera, a fruit fly species used in this article</td>
		</tr>
		<tr>
			<td>Plastic vial</td>
			<td>Hightech</td>
			<td>MKC-30</td>
			<td>Plastic vial, for fly stock maintenance</td>
		</tr>
		<tr>
			<td>Buzz plugs vial and bottle closures for glass vials</td>
			<td>Fisher Scientific</td>
			<td>AS-271</td>
			<td>Cellulose plug, for fly stock maintenance</td>
		</tr>
		<tr>
			<td>White soft sugar</td>
			<td>Mitsui Sugar</td>
			<td>J-500g</td>
			<td>White soft sugar, for standard cornmeal/sugar/yeast/agar food</td>
		</tr>
		<tr>
			<td>Corn flour</td>
			<td>Nippon Flour Mills</td>
			<td>F</td>
			<td>Corn flour, for standard cornmeal/sugar/yeast/agar food</td>
		</tr>
		<tr>
			<td>Corn grits - C</td>
			<td>Nippon Flour Mills</td>
			<td>GC</td>
			<td>Corn grits - C, for standard cornmeal/sugar/yeast/agar food</td>
		</tr>
		<tr>
			<td>Agar powder</td>
			<td>Matsuki Kanten Sangyo</td>
			<td>No.602</td>
			<td>Agar powder, for standard cornmeal/sugar/yeast/agar food</td>
		</tr>
		<tr>
			<td>Dry beer yeast</td>
			<td>Asahi Food &#38; Healthcare</td>
			<td>Y2A</td>
			<td>Dry beer yeast, for standard cornmeal/sugar/yeast/agar food</td>
		</tr>
		<tr>
			<td>Butyl p-hydroxybenzoate</td>
			<td>Nacalai Tesque</td>
			<td>06327-02</td>
			<td>Butyl p-hydroxybenzoate, for standard cornmeal/sugar/yeast/agar food</td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Wako</td>
			<td>057-00456</td>
			<td>Ethanol, for standard cornmeal/sugar/yeast/agar food</td>
		</tr>
		<tr>
			<td>Flat bottom microtube</td>
			<td>Ina Optica</td>
			<td>CF-0150</td>
			<td>1.5 mL microtube, for collecting pupae</td>
		</tr>
		<tr>
			<td>CAPSULEFUGE</td>
			<td>Tomy</td>
			<td>PMC-060</td>
			<td>Mini microcentrifuge, for collecting pupae</td>
		</tr>
		<tr>
			<td>Sterilized Schale NB</td>
			<td>Sansei Medical</td>
			<td>01-013</td>
			<td>Plastic Petri dish (diameter 90 mm x height 15 mm)</td>
		</tr>
		<tr>
			<td>Serum tube rack</td>
			<td>Iwaki</td>
			<td>9796-050</td>
			<td>Used as a moist chamber, for observation of pupa</td>
		</tr>
		<tr>
			<td>Corning Falcon Easy-Grip tissue culture dish</td>
			<td>Corning</td>
			<td>353001</td>
			<td>Plastic Petri dish (diameter 35 mm x height 10 mm)</td>
		</tr>
		<tr>
			<td>Falcon standard tissue culture dish</td>
			<td>Corning</td>
			<td>353002</td>
			<td>Plastic Petri dish (diameter 60 mm x height 15 mm)</td>
		</tr>
		<tr>
			<td>Push-pin</td>
			<td>Kokuyo</td>
			<td>51233709</td>
			<td>Push-pin, for making pinholes on the microtube lid</td>
		</tr>
		<tr>
			<td>Stereomicroscope</td>
			<td>Olympus</td>
			<td>SZX16</td>
			<td>Stereomicroscope, for morphological observation</td>
		</tr>
		<tr>
			<td>Digital camera</td>
			<td>Olympus</td>
			<td>DSE-330-A</td>
			<td>Digital camera, for imaging</td>
		</tr>
		<tr>
			<td>NICETACK double sided tape</td>
			<td>Nichiban</td>
			<td>NW-15SF</td>
			<td>Double sided tape, for removing puparium</td>
		</tr>
		<tr>
			<td>Dumont #5 forceps</td>
			<td>Fine Science Tools</td>
			<td>11252-20</td>
			<td>Forceps, for removing puparium</td>
		</tr>
		<tr>
			<td>Van Gogh VISUAL Paint brush</td>
			<td>Talens Japan</td>
			<td>GWVR-#5/0</td>
			<td>Paint brush, for removing puparium</td>
		</tr>
		<tr>
			<td>Greiner CELLSTAR 12 well cell culture plate</td>
			<td>Merck</td>
			<td>665-180</td>
			<td>12-well cell culture plate, for measuring durations of pupal periods</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Wako</td>
			<td>191-01665</td>
			<td>NaCl, for PBS</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Nacalai Tesque</td>
			<td>285-14</td>
			<td>KCl, for PBS</td>
		</tr>
		<tr>
			<td>Na2HPO4&#183;12H2O</td>
			<td>Wako</td>
			<td>196-02835</td>
			<td>Na2HPO4&#183;12H2O, for PBS</td>
		</tr>
		<tr>
			<td>KH2PO4</td>
			<td>Nacalai Tesque</td>
			<td>28721-55</td>
			<td>KH2PO4, for PBS</td>
		</tr>
		<tr>
			<td>Stepped Neutral Density (ND) Filter 0.04 - 3.0</td>
			<td>Edmund Optics</td>
			<td>64-384</td>
			<td>Stepped density filter, for calibration of pigmentation measurement</td>
		</tr>
		<tr>
			<td>ImageJ software</td>
			<td>NIH</td>
			<td>1.8.0-101</td>
			<td>ImageJ software, for measurement of intensity of black spots on a wing (https://imagej.nih.gov)</td>
		</tr>
		<tr>
			<td>FINE FROST glass slide</td>
			<td>Matsunami Glass Ind</td>
			<td>FF-001</td>
			<td>Glass slide, for measurement of intensity of black spots on a wing</td>
		</tr>
		<tr>
			<td>Square microscope cover glass 18 x 18</td>
			<td>Matsunami Glass Ind</td>
			<td>C018181</td>
			<td>Cover slip, for measurement of intensity of black spots on a wing</td>
		</tr>
	</tbody>
</table>

methods for staging pupal periods and measurement of wing pigmentation of drosophila guttifera

다양 한 종의 초파리 (과일 파리) 개발 및 유전자 변화 진화 변화에 대 한 책임의 메커니즘을 연구 하는 기회를 제공 합니다. 특히, 성인 무대 날개 착 색 비교를 포함 하 여 interspecific 비교 형태학 상 특성의 풍부한 원천입니다. 공부 하 고 종 간의 발달 차이, 자세한 관찰과 적절 한 준비는 정확한 비교 필요 합니다. 여기 우리가 번데기 기간의 준비 및 폴카 점 과일 파리, 초파리 guttifera착 색 날개의 정량화에 대 한 프로토콜을 설명합니다. 첫째, 자세한 형태 관찰 및 형태학에 따라 번데기 단계의 정의 방법을 설명 합니다. 이 방법은 외부 chitinous 사건의 번데기, 번데기 형태학의 상세한 관측을 사용 하는 puparium를 제거 하기 위한 기술을 포함 합니다. 둘째, 우리는 정의 된 번데기 단계 기간을 측정 하기 위한 방법을 설명 합니다. 마지막으로, 디지털 이미지 및 ImageJ 소프트웨어를 사용 하 여 이미지 분석에 따라 날개 염색의 정량화에 대 한 방법을 설명 합니다. 이러한 방법으로, 우리는 번데기 단계 동안 성인 특성의 발달 과정을 비교 하기 위한 견고한 기초를 설정할 수 있습니다.

D. guttifera 의 번데기 기간 17 단계 (P1-P15(ii), 단계 (P1, P5-6, P10) 그림 3, 그리고 모든 17 단계는 그림 4에 나와 있는 표시 됩니다 3 명의 대표자의 이미지)으로 나누어져 있습니다. 베인 브리지 및 Bownes17 20 단계 D. melanogaster인정, 이러한 단계 중 일부 수 하지에 적용할 D. guttifera. 2 개의 발달 이벤트의 순서, 노란 몸 (midgut26내 창 고 세포의 질량)의 모양 및 녹색, 터 닝 말 tubules의 타이밍 제어 되지 않습니다 엄격 하 게 D. guttifera에 그리고 그러므로 우리가 P5를 분리할 수 없었다 (i ), P5(ii) 및 P6. 또한, 달리 D. melanogaster, 흉부 및 복 부 bristles의 흑의 타이밍은 동기화, 그리고 그러므로 우리 P11(i)과 P11(ii)19를 분리할 수 없었다.
우리 디 guttifera (Fukutomi 외.19에서 표 3)의 번데기 단계의 길이 측정할 수 있습니다. 전체 번데기 기간은 약 20 h 25 ° C17에 D. melanogaster 의 그것 보다 더 이상 이다. 우리는 주변 지역 campaniform sensillum, 경도 정 맥 팁 그리고 후부 crossvein의 ΔODs를 계산합니다. 여기, 우리가 보여줍니다 ODs 및 ΔODs 성인 일수로 (표 4) 후 7 일. 여러 단계의 데이터를 비교 하 여 우리는 발견 단계 P12(i) 착 색의 개시의 타이밍은 그 염색 일수로 후 24 h에 의해 완료 됩니다 (그림 5, Fukutomi 외에 사용 되는 원래 치수에서. 19).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56935/56935fig1.jpg"> 그림 1입니다. 그림 puparium 제거. (A) 번데기 복 부 측 이중 면 테이프의 조각의를 놓습니다. puparium의 앞쪽 부분을 제거 합니다. (B)는 puparium 복 부 측에서 집게와 휴식. (C)는 puparium를 해제 후 그림 붓을 사용 하 여 번데기를 꺼내. <a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56935/56935fig1large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭 하십시오.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56935/56935fig2.jpg"> 그림 2입니다. 착 색을 측정 하기 위한 영역의 정의. (Campaniform sensillum와 관련 된 자리 A) 점 A. (B) 지점 B 경도 정 맥 팁과 관련 된 장소에 대 한. (C) 지점 C 후부 crossvein와 관련 된 자리. (D) 포인트 D 제어 영역에 대 한. 눈금 막대 표시 250 µ m. <a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56935/56935fig2large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭 하십시오.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56935/56935fig3.jpg"> 그림 3입니다. 정의 된 번데기 단계의 예. (A) 번데기 단계의 P1 puparium 덮여. (B) 단계 P5-6의 번데기. (C) 단계 P10의 번데기입니다. (B)에서 관찰 하기 전에 puparia 제거 및 (C). 눈금 막대 표시 500 µ m. <a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56935/56935fig3large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭 하십시오.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/56935/56935fig4.jpg"> 그림 4입니다. D. guttifera에서 확인 된 17 번데기 단계의 그림. (A) P1에 puparium 흰색입니다. (B) P2는 puparium의 색상은 갈색 빛 이다. (C) P3, 거품 측면 사이드 (검은 화살표)에서 관찰 됩니다. (D) P4(i), 거품 P3 보다 크면 (검은 화살표), 번데기는 PBS에 부 력. (E) P4(ii), 간격 앞쪽 부분 (검은 화살표)와 후부 부분 (회색 화살촉)에서 관찰 됩니다. (F) p 5-6, tubules 마이그레이션 (puparium에 의해 덮여 있는지 어려운) 하는 말 번데기 모양은 번데기 상피와 번데기 표 피에 의해 형성 된다. (G) P7, 노란 몸 등 쪽 측 (검은 화살표)에서 관찰할 수 있습니다. (H) P8, 눈은 노란색. (I) P9, 눈은 주황색. (J) P10, 눈은 빨간색. (K) P11, 궤도 및 ocellar bristles (회색 화살촉), 털, 흉부 macrochaetae (검은 화살표), 그리고 tarsal bristles 검 고 볼 수 있습니다. (L) P12(i), 날개의 팁은 회색. (M) P12(ii), 날개의 모든 부분에는 회색 (검은 화살표)입니다. (N) P13, 날개는 완전히 검은색. (P14 O), 머리와 다리는 완전히 어둡게 된다. (P) P15(i)는 meconium 등 쪽 복 부 (검은 화살표)에 관찰할 수 있습니다. (Q) P15(ii), 비행 eclosing입니다. 이 단계의 정보는 Fukutomi 외에 설명 했다. 19 <a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56935/56935fig4large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭 하십시오.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/56935/56935fig5.jpg"> 그림 5입니다. 날개에 companiform sensillum 주위 착 색의 개발. 서클 개별 ΔODs 나타내고 가로 막대 평균을 표시 합니다. P10: n = 10, P11: n = 10, 12(i): n = 10, P15(i): n = 11, 3 h: n = 8, 24 h: n = 7, 7 일: n = 10. <a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56935/56935fig5large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭 하십시오.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td>구성 요소</td>
			<td></td>
		</tr>
<tr>
<td>화이트 소프트 설탕</td>
			<td>51.6 g</td>
		</tr>
<tr>
<td>옥수수 가루</td>
			<td>172.4 g</td>
		</tr>
<tr>
<td>옥수수 밀가루-C</td>
			<td>86.4 g</td>
		</tr>
<tr>
<td>건조 맥주 효 모</td>
			<td>106 g</td>
		</tr>
<tr>
<td>한 천 분말</td>
			<td>35.28 g</td>
		</tr>
<tr>
<td>ddH2O</td>
			<td>mL 4000</td>
		</tr>
<tr>
<td colspan="2">30 분 및 70 ° c.에 아래로 냉각을 위한 종</td>
		</tr>
<tr>
<td colspan="2">추가 4 g 부 틸 p-hydroxybenzoate의 에탄올 40 mL에 용 해.</td>
		</tr>
<tr>
<td colspan="2">잘 혼합 하 고 플라스틱 튜브 9 mL를 붓고 (직경 25 m m x 높이 96 m m).</td>
		</tr>
</tbody></table>표 1입니다. 표준 옥수수 가루/설탕/효 모/천 식품의 구성.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td>구성 요소</td>
			<td>금액</td>
		</tr>
<tr>
<td>NaCl</td>
			<td>80 g</td>
		</tr>
<tr>
<td>KCl</td>
			<td>2 세대</td>
		</tr>
<tr>
<td>나2HPO4·12H2O</td>
			<td>29 g</td>
		</tr>
<tr>
<td>KH2포4
</td>
			<td>2 세대</td>
		</tr>
<tr>
<td>ddH2O</td>
			<td>최대 10 L</td>
		</tr>
<tr>
<td></td>
			<td>pH 7.4를 설정</td>
		</tr>
</tbody></table>표 2입니다. PBS (X 1) 구성입니다.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td>단계</td>
			<td>시간 (h)의 의미</td>
			<td>사 우 스 다코타</td>
			<td>n</td>
		</tr>
<tr>
<td>P1-2</td>
			<td>1.7</td>
			<td>0.65</td>
			<td>16</td>
		</tr>
<tr>
<td>P3</td>
			<td>2.1</td>
			<td>0.65</td>
			<td>16</td>
		</tr>
<tr>
<td>P4(i)</td>
			<td>2.1</td>
			<td>1.69</td>
			<td>19</td>
		</tr>
<tr>
<td>P4(ii)</td>
			<td>0.3</td>
			<td>0.28</td>
			<td>29</td>
		</tr>
<tr>
<td>P5-6</td>
			<td>5.0</td>
			<td>3.07</td>
			<td>30</td>
		</tr>
<tr>
<td>P7</td>
			<td>31.9</td>
			<td>7.22</td>
			<td>46</td>
		</tr>
<tr>
<td>P8</td>
			<td>9.6</td>
			<td>2.81</td>
			<td>57</td>
		</tr>
<tr>
<td>P9</td>
			<td>10.9</td>
			<td>2.66</td>
			<td>55</td>
		</tr>
<tr>
<td>P10</td>
			<td>11.7</td>
			<td>2.96</td>
			<td>39</td>
		</tr>
<tr>
<td>P11</td>
			<td>4.4</td>
			<td>2.81</td>
			<td>44</td>
		</tr>
<tr>
<td>P12(i)</td>
			<td>1.1</td>
			<td>0.76</td>
			<td>44</td>
		</tr>
<tr>
<td>P12(ii)</td>
			<td>2.0</td>
			<td>0.70</td>
			<td>43</td>
		</tr>
<tr>
<td>P13</td>
			<td>2.2</td>
			<td>0.68</td>
			<td>10</td>
		</tr>
<tr>
<td>P14-15(i)</td>
			<td>28.6</td>
			<td>2.75</td>
			<td>10</td>
		</tr>
<tr>
<td>P15(ii)</td>
			<td>1.4</td>
			<td>0.87</td>
			<td>10</td>
		</tr>
<tr>
<td>총</td>
			<td>121.7</td>
			<td></td>
			<td></td>
		</tr>
</tbody></table>테이블 3입니다. 측정의 번데기 단계의 기간 D. guttifera.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td></td>
			<td>OD</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td>ΔOD</td>
			<td></td>
			<td></td>
		</tr>
<tr>
<td></td>
			<td>Campaniform sensilum</td>
			<td>경도 정 맥 팁</td>
			<td>후부 crossvein</td>
			<td>제어</td>
			<td></td>
			<td>Campaniform sensilum</td>
			<td>경도 정 맥 팁</td>
			<td>후부 crossvein</td>
		</tr>
<tr>
<td>개인</td>
			<td>(A)</td>
			<td>(B)</td>
			<td>(C 지점)</td>
			<td>(포인트 D)</td>
			<td></td>
			<td>(A-포인트 D)</td>
			<td>(B-포인트 D)</td>
			<td>(포인트 C-포인트 D)</td>
		</tr>
<tr>
<td>1</td>
			<td>0.549</td>
			<td>0.484</td>
			<td>0.515</td>
			<td>0.256</td>
			<td></td>
			<td>0.293</td>
			<td>0.228</td>
			<td>0.259</td>
		</tr>
<tr>
<td>2</td>
			<td>0.529</td>
			<td>0.489</td>
			<td>0.516</td>
			<td>0.254</td>
			<td></td>
			<td>0.275</td>
			<td>0.235</td>
			<td>0.262</td>
		</tr>
<tr>
<td>3</td>
			<td>0.546</td>
			<td>0.48</td>
			<td>0.533</td>
			<td>0.255</td>
			<td></td>
			<td>0.291</td>
			<td>0.225</td>
			<td>0.278</td>
		</tr>
<tr>
<td>4</td>
			<td>0.583</td>
			<td>0.496</td>
			<td>0.566</td>
			<td>0.255</td>
			<td></td>
			<td>0.328</td>
			<td>0.241</td>
			<td>0.311</td>
		</tr>
<tr>
<td>5</td>
			<td>0.523</td>
			<td>0.479</td>
			<td>0.528</td>
			<td>0.235</td>
			<td></td>
			<td>0.288</td>
			<td>0.244</td>
			<td>0.293</td>
		</tr>
<tr>
<td>6</td>
			<td>0.572</td>
			<td>0.509</td>
			<td>0.546</td>
			<td>0.265</td>
			<td></td>
			<td>0.307</td>
			<td>0.244</td>
			<td>0.281</td>
		</tr>
<tr>
<td>7</td>
			<td>0.568</td>
			<td>0.511</td>
			<td>0.56</td>
			<td>0.256</td>
			<td></td>
			<td>0.312</td>
			<td>0.255</td>
			<td>0.304</td>
		</tr>
<tr>
<td>8</td>
			<td>0.56</td>
			<td>0.507</td>
			<td>0.562</td>
			<td>0.27</td>
			<td></td>
			<td>0.29</td>
			<td>0.237</td>
			<td>0.292</td>
		</tr>
<tr>
<td>9</td>
			<td>0.551</td>
			<td>0.485</td>
			<td>0.569</td>
			<td>0.259</td>
			<td></td>
			<td>0.292</td>
			<td>0.226</td>
			<td>0.31</td>
		</tr>
</tbody></table>표 4입니다. ODs를 측정 하 고 ΔODs D. guttifera 성인 일수로 후 7 일.

Watch this Scientific Journal Video about Methods for Staging Pupal Periods and Measurement of Wing Pigmentation of Drosophila guttifera at JoVE.com

Methods for Staging Pupal Periods and Measurement of Wing Pigmentation of Drosophila guttifera

번데기 기간 및 측정 초파리 guttifera 의 날개 염색의 준비에 대 한 프로토콜을 설명 합니다. 준비와 염색의 정량화 성인 특성의 발달 기계 장치를 공부에 대 한 견고한 기초를 제공 하 고 특성 개발의 interspecific 비교 사용.

준비 번데기 기간 및 초파리 guttifera 의 날개 색소 측정 방법

Diversified species of Drosophila (fruit fly) provide opportunities to study mechanisms of development and genetic changes responsible for evolutionary ...

methods-for-staging-pupal-periods-measurement-wing-pigmentation

Nanyang Technological University

Research

JoVE Journal

Biology

Methods for Staging Pupal Periods and Measurement of Wing Pigmentation of Drosophila guttifera

11.5K 조회수.  Kyoto University. 번데기 기간 및 측정 초파리 guttifera 의 날개 염색의 준비에 대 한 프로토콜을 설명 합니다. 준비와 염색의 정량화 성인 특성의 발달 기계 장치를 공부에 대 한 견고한 기초를 제공 하 고 특성 개발의 interspecific 비교 사용.

비디오: Methods for Staging Pupal Periods and Measurement of Wing Pigmentation of Drosophila guttifera

High-resolution Measurement of Odor-Driven Behavior in Drosophila Larvae

Olfactory responses in Drosophila larvae have been traditionally studied in Petri dishes comprising a single peripheral odor source. In this behavioral paradigm, the experimenter usually assumes that the rapid diffusion of odorant molecules from the source leads to the creation of a stable gradient in the dish. To establish a quantitative correlation between sensory inputs and behavioral responses, it is necessary to achieve a more thorough characterization of the odorant stimulus conditions. In this video article, we describe a new method allowing the construction of odorant gradients with stable and controllable geometries. We briefly illustrate how these gradients can be used to screen for olfactory defects (full and partial anosmia) and to study more subtle features of chemotaxis behavior.

In this video article, we describe a new method allowing the construction of odorant gradients with stable and controllable geometries. We briefly illustrate how these gradients can be used to screen for olfactory defects (full and partial anosmia) and to study more subtle features of chemotaxis behavior.

Drosophila의 유충에서 냄새 기반 행동의 고해상도 측정

Olfactory responses in Drosophila larvae have been traditionally studied in Petri dishes comprising a single peripheral odor source. In this behavioral ...

연구

생물학

Electrophysiological Methods for Recording Synaptic Potentials from the NMJ of Drosophila Larvae

In this video, we describe the electrophysiological methods for recording synaptic transmission at the neuromuscular junction (NMJ) of Drosophila larva. The larval neuromuscular system is a model synapse for the study of synaptic physiology and neurotransmission, and is a valuable research tool that has defined genetics and is accessible to experimental manipulation. Larvae can be dissected to expose the body wall musculature, central nervous system, and peripheral nerves. The muscles of Drosophila and their innervation pattern are well characterized and muscles are easy to access for intracellular recording. Individual muscles can be identified by their location and orientation within the 8 abdominal segments, each with 30 muscles arranged in a pattern that is repeated in segments A2 - A7. Dissected drosophila larvae are thin and individual muscles and bundles of motor neuron axons can be visualized by transillumination1. Transgenic constructs can be used to label target cells for visual identification or for manipulating gene products in specific tissues. In larvae, excitatory junction potentials (EJP&#8217;s) are generated in response to vesicular release of glutamate from the motoneurons at the synapse. In dissected larvae, the EJP can be recorded in the muscle with an intracellular electrode. Action potentials can be artificially evoked in motor neurons that have been cut posterior to the ventral ganglion, drawn into a glass pipette by gentle suction and stimulated with an electrode. These motor neurons have distinct firing thresholds when stimulated, and when they fire simultaneously, they generate a response in the muscle. Signals transmitted across the NMJ synapse can be recorded in the muscles that the motor neurons innervate. The EJP&#8217;s and minature excitatory junction potentials (mEJP&#8217;s) are seen as changes in membrane potential. Electrophysiological responses are recorded at room temperature in modified minimal hemolymph-like solution2 (HL3) that contains 5 mM Mg2+ and 1.5 mM Ca2+. Changes in the amplitude of evoked EJP&#8217;s can indicate differences in synaptic function and structure. Digitized recordings are analyzed for EJP amplitude, mEJP frequency and amplitude, and quantal content.

Here we describe electrophysiological methods for measuring synaptic transmission at the neuromuscular junction of Drosophila larva. Evoked release is initiated artificially by stimulating the motor neuron axons, and transmission through the NMJ can be measured by the postsynaptic response evoked in the muscle.

Drosophila의 애벌레의 NMJ에서 시냅틱 후보를 녹음 Electrophysiological 방법

In this video, we describe the electrophysiological methods for recording synaptic transmission at the neuromuscular junction (NMJ) of Drosophila larva. ...

Live Dissection of Drosophila Embryos: Streamlined Methods for Screening Mutant Collections by Antibody Staining

Drosophila embryos between stages 14 and 17 of embryonic development can be readily dissected to generate "fillet" preparations. In these preparations, the central nervous system runs down the middle, and is flanked by the body walls. Many different phenotypes have been examined using such preparations. In most cases, the fillets were generated by dissection of antibody-stained fixed whole-mount embryos. These "fixed dissections" have some disadvantages, however. They are time-consuming to execute, and it is difficult to sort mutant (GFP-negative) embryos from stocks in which mutations are maintained over GFP balancer chromosomes. Since 2002, our group has been conducting deficiency and ectopic expression screens to identify ligands for orphan receptors. In order to do this, we developed streamlined protocols for live embryo dissection and antibody staining of collections containing hundreds of balanced lines. We have concluded that it is considerably more efficient to examine phenotypes in large collections of stocks by live dissection than by fixed dissection. Using the protocol described here, a single trained individual can screen up to 10 lines per day for phenotypes, examining 4-7 mutant embryos from each line under a compound microscope. This allows the identification of mutations conferring subtle, low-penetrance phenotypes, since up to 70 hemisegments per line are scored at high magnification with a 40X water-immersion lens.

Live Dissection of Drosophila Embryos: Streamlined Methods for Screening Mutant Collections by Antibody Staining

We describe a streamlined protocol for generating "fillet" preparations of Drosophila embryos of specific genotypes. This protocol allows efficient execution of a variety of genetic screens. It also allows excellent visualization of structures in the late embryo.

라이브 해부 Drosophila 태아 : 항체 스테 이닝에 의해 돌연변이 컬렉션을 검사 늘씬한 방법

Drosophila embryos between stages 14 and 17 of embryonic development can be readily dissected to generate "fillet" preparations.  In these preparations, ...

Laser Microdissection Applied to Gene Expression Profiling of Subset of Cells from the Drosophila Wing Disc

Heterogeneous nature of tissues has proven to be a limiting factor in the amount of information that can be generated from biological samples, compromising downstream analyses. Considering the complex and dynamic cellular associations existing within many tissues, in order to recapitulate the in vivo interactions thorough molecular analysis one must be able to analyze specific cell populations within their native context. Laser-mediated microdissection can achieve this goal, allowing unambiguous identification and successful harvest of cells of interest under direct microscopic visualization while maintaining molecular integrity. We have applied this technology to analyse gene expression within defined areas of the developing Drosophila wing disc, which represents an advantageous model system to study growth control, cell differentiation and organogenesis. Larval imaginal discs are precociously subdivided into anterior and posterior, dorsal and ventral compartments by lineage restriction boundaries. Making use of the inducible GAL4-UAS binary expression system, each of these compartments can be specifically labelled in transgenic flies expressing an UAS-GFP transgene under the control of the appropriate GAL4-driver construct. In the transgenic discs, gene expression profiling of discrete subsets of cells can precisely be determined after laser-mediated microdissection, using the fluorescent GFP signal to guide laser cut. 
Among the variety of downstream applications, we focused on RNA transcript profiling after localised RNA interference (RNAi). With the advent of RNAi technology, GFP labelling can be coupled with localised knockdown of a given gene, allowing to determinate the transcriptional response of a discrete cell population to the specific gene silencing. To validate this approach, we dissected equivalent areas of the disc from the posterior (labelled by GFP expression), and the anterior (unlabelled) compartment upon regional silencing in the P compartment of an otherwise ubiquitously expressed gene. RNA was extracted from microdissected silenced and unsilenced areas and comparative gene expression profiling determined by quantitative real-time RT-PCR. We show that this method can effectively be applied for accurate transcriptomics of subsets of cells within the Drosophila imaginal discs. Indeed, while massive disc preparation as source of RNA generally assumes cell homogeneity, it is well known that transcriptional expression can vary greatly within these structures in consequence of positional information. Using localized fluorescent GFP signal to guide laser cut, more accurate transcriptional analyses can be performed and profitably applied to disparate applications, including transcript profiling of distinct cell lineages within their native context.

Laser Microdissection Applied to Gene Expression Profiling of Subset of Cells from the Drosophila Wing Disc

Laser microdissection was applied to analyse gene expression profiling in specific compartments of Drosophila wing disc subjected to localised RNAi in vivo. RNA extracted from equivalent areas of silenced and unsilenced compartments was analysed by quantitative RT-PCR to determine comparative gene expression profiling within the context of native tissue microecology.

레이저 Microdissection는 세포의 하위 집합 유전자 발현 프로 파일링에 적용 Drosophila 윙 디스크

Heterogeneous nature of tissues has proven to be a limiting factor in the amount of information that can be generated from biological samples, ...

Drosophila Pupal Abdomen Immunohistochemistry

The Drosophila pupal abdomen is an established model system for the study of epithelial morphogenesis and the development of sexually dimorphic morphologies 1-3. During pupation, which spans approximately 96 hours (at 25 &deg;C), proliferating populations of imaginal cells replace the larval epidermis to generate the adult abdominal segments. These imaginal cells, born during embryogenesis, exist as lateral pairs of histoblast nests in each abdominal segment of the larvae. Four pairs of histoblast nests give rise to the adult dorsal cuticle (anterior and posterior dorsal nests), the ventral cuticle (ventral nests) and the spiracles associated with each segment (spiracle nests) 4. Upon puparation, these diploid cells (distinguishable by size from the larger polyploid larval epidermal cells- LECs) begin a stereotypical process of proliferation, migration and replacement of the LECs. Various molecular and genetic tools can be employed to investigate the contributions of genetic pathways involved in morphogenesis of the adult abdomen. Ultimate adult phenotypes are typically analyzed following dissection of adult abdominal cuticles. However, investigation of the underlying molecular processes requires immunohistochemical analyses of the pupal epithelium, which present unique challenges. Temporally dynamic morphogenesis and the interactions of two distinct epithelial populations (larval and imaginal) generate a fragile tissue prone to excessive cell loss during dissection and subsequent processing. We have developed methods of dissection, fixation, mounting and imaging of the Drosophila pupal abdominem epithelium for immunohistochemical studies that generate consistent high quality samples suitable for confocal or standard fluorescent microscopy.

Drosophila Pupal Abdomen Immunohistochemistry

Antibody staining of the Drosophila pupae can enhance genetic analyses of adult abdominal developmental genetics. We present our protocol for dissection, fixation and antibody staining of staged Drosophila pupal abdomen.

Drosophila Pupal의 복부 Immunohistochemistry

The Drosophila pupal abdomen is an established model system for the study of epithelial morphogenesis and the development of sexually dimorphic ...

Measurement of Lifespan in Drosophila melanogaster

Aging is a phenomenon that results in steady physiological deterioration in nearly all organisms in which it has been examined, leading to reduced physical performance and increased risk of disease. Individual aging is manifest at the population level as an increase in age-dependent mortality, which is often measured in the laboratory by observing lifespan in large cohorts of age-matched individuals. Experiments that seek to quantify the extent to which genetic or environmental manipulations impact lifespan in simple model organisms have been remarkably successful for understanding the aspects of aging that are conserved across taxa and for inspiring new strategies for extending lifespan and preventing age-associated disease in mammals.
The vinegar fly, Drosophila melanogaster, is an attractive model organism for studying the mechanisms of aging due to its relatively short lifespan, convenient husbandry, and facile genetics. However, demographic measures of aging, including age-specific survival and mortality, are extraordinarily susceptible to even minor variations in experimental design and environment, and the maintenance of strict laboratory practices for the duration of aging experiments is required. These considerations, together with the need to practice careful control of genetic background, are essential for generating robust measurements. Indeed, there are many notable controversies surrounding inference from longevity experiments in yeast, worms, flies and mice that have been traced to environmental or genetic artifacts1-4. In this protocol, we describe a set of procedures that have been optimized over many years of measuring longevity in Drosophila using laboratory vials. We also describe the use of the dLife software, which was developed by our laboratory and is available for download (<a href="http://sitemaker.umich.edu/pletcherlab/software">http://sitemaker.umich.edu/pletcherlab/software</a>). dLife accelerates throughput and promotes good practices by incorporating optimal experimental design, simplifying fly handling and data collection, and standardizing data analysis. We will also discuss the many potential pitfalls in the design, collection, and interpretation of lifespan data, and we provide steps to avoid these dangers.

Measurement of Lifespan in Drosophila melanogaster

Drosophila melanogaster is a powerful model organism for exploring the molecular basis of longevity regulation. This protocol will discuss the steps involved in generating a reproducible, population-based measurement of longevity as well as potential pitfalls and how to avoid them.

의 수명 측정<em&gt; Drosophila melanogaster</em

Aging is a phenomenon that results in steady physiological deterioration in nearly all organisms in which it has been examined, leading to reduced ...

An Improved Method for Accurate and Rapid Measurement of Flight Performance in Drosophila

Drosophila has proven to be a useful model system for analysis of behavior, including flight. The initial flight tester involved dropping flies into an oil-coated graduated cylinder; landing height provided a measure of flight performance by assessing how far flies will fall before producing enough thrust to make contact with the wall of the cylinder. Here we describe an updated version of the flight tester with four major improvements. First, we added a &#34;drop tube&#34; to ensure that all flies enter the flight cylinder at a similar velocity between trials, eliminating variability between users. Second, we replaced the oil coating with removable plastic sheets coated in Tangle-Trap, an adhesive designed to capture live insects. Third, we use a longer cylinder to enable more accurate discrimination of flight ability. Fourth we use a digital camera and imaging software to automate the scoring of flight performance. These improvements allow for the rapid, quantitative assessment of flight behavior, useful for large datasets and large-scale genetic screens.

An Improved Method for Accurate and Rapid Measurement of Flight Performance in Drosophila

Here we describe a method for rapid and accurate measurement of flight performance in Drosophila, enabling high-throughput screening.

비행 성능의 정확하고 빠른 측정을위한 개선 된 방법에<em&gt; 초파리</em

Drosophila has proven to be a useful model system for analysis of behavior, including flight. The initial flight tester involved dropping flies into an ...

Imaging Through the Pupal Case of Drosophila melanogaster

The longstanding use of Drosophila as a model for cell and developmental biology has yielded an array of tools. Together, these techniques have enabled analysis of cell and developmental biology from a variety of methodological angles. Live imaging is an emerging method for observing dynamic cell processes, such as cell division or cell motility. Having isolated mutations in uncharacterized putative cell cycle proteins it became essential to observe mitosis in situ using live imaging. Most live imaging studies in Drosophila have focused on the embryonic stages that are accessible to manipulation and observation because of their small size and optical clarity. However, in these stages the cell cycle is unusual in that it lacks one or both of the gap phases. By contrast, cells of the pupal wing of Drosophila have a typical cell cycle and undergo a period of rapid mitosis spanning about 20 hr of pupal development. It is easy to identify and isolate pupae of the appropriate stage to catch mitosis in situ. Mounting intact pupae provided the best combination of tractability and durability during imaging, allowing experiments to run for several hours with minimal impact on cell and animal viability. The method allows observation of features as small as, or smaller than, fly chromosomes. Adjustment of microscope settings and the details of mounting, allowed extension of the preparation to visualize membrane dynamics of adjacent cells and fluorescently labeled proteins such as tubulin. This method works for all tested fluorescent proteins and can capture submicron scale features over a variety of time scales. While limited to the outer 20 &#181;m of the pupa with a conventional confocal microscope, this approach to observing protein and cellular dynamics in pupal tissues in vivo may be generally useful in the study of cell and developmental biology in these tissues.

Imaging Through the Pupal Case of Drosophila melanogaster

This paper demonstrates the use of a fast scanning confocal microscope to image cell behavior directly through the puparium. By leaving the pupal case intact, this method allows observation and measurement of dynamic cell processes at a stage of Drosophila development that is difficult to study directly.

의 번데기 케이스를 통해서 영상<em&gt; 초파리</em

The longstanding use of Drosophila as a model for cell and developmental biology has yielded an array of tools. Together, these techniques have enabled ...

Measurement of Metabolic Rate in Drosophila using Respirometry

Metabolic disorders are a frequent problem affecting human health. Therefore, understanding the mechanisms that regulate metabolism is a crucial scientific task. Many disease causing genes in humans have a fly homologue, making Drosophila a good model to study signaling pathways involved in the development of different disorders. Additionally, the tractability of Drosophila simplifies genetic screens to aid in identifying novel therapeutic targets that may regulate metabolism. In order to perform such a screen a simple and fast method to identify changes in the metabolic state of flies is necessary. In general, carbon dioxide production is a good indicator of substrate oxidation and energy expenditure providing information about metabolic state. In this protocol we introduce a simple method to measure CO2 output from flies. This technique can potentially aid in the identification of genetic perturbations affecting metabolic rate.

Measurement of Metabolic Rate in Drosophila using Respirometry

Metabolic disorders are among one of the most common diseases in humans. The genetically tractable model organism D. melanogaster can be used to identify novel genes that regulate metabolism. This paper describes a relatively simple method which allows studying the metabolic rate in flies by measuring their CO2 production.

대사율의 측정에<em&gt; 초파리</em&gt; Respirometry를 사용하여

Metabolic disorders are a frequent problem affecting human health. Therefore, understanding the mechanisms that regulate metabolism is a crucial ...

Ex vivo Culture of Drosophila Pupal Testis and Single Male Germ-line Cysts: Dissection, Imaging, and Pharmacological Treatment

During spermatogenesis in mammals and in Drosophila melanogaster, male germ cells develop in a series of essential developmental processes. This includes differentiation from a stem cell population, mitotic amplification, and meiosis. In addition, post-meiotic germ cells undergo a dramatic morphological reshaping process as well as a global epigenetic reconfiguration of the germ line chromatin&#8212;the histone-to-protamine switch.
Studying the role of a protein in post-meiotic spermatogenesis using mutagenesis or other genetic tools is often impeded by essential embryonic, pre-meiotic, or meiotic functions of the protein under investigation. The post-meiotic phenotype of a mutant of such a protein could be obscured through an earlier developmental block, or the interpretation of the phenotype could be complicated. The model organism Drosophila melanogaster offers a bypass to this problem: intact testes and even cysts of germ cells dissected from early pupae are able to develop ex vivo in culture medium. Making use of such cultures allows microscopic imaging of living germ cells in testes and of germ-line cysts. Importantly, the cultivated testes and germ cells also become accessible to pharmacological inhibitors, thereby permitting manipulation of enzymatic functions during spermatogenesis, including post-meiotic stages.
The protocol presented describes how to dissect and cultivate pupal testes and germ-line cysts. Information on the development of pupal testes and culture conditions are provided alongside microscope imaging data of live testes and germ-line cysts in culture. We also describe a pharmacological assay to study post-meiotic spermatogenesis, exemplified by an assay targeting the histone-to-protamine switch using the histone acetyltransferase inhibitor anacardic acid. In principle, this cultivation method could be adapted to address many other research questions in pre- and post-meiotic spermatogenesis.

Ex vivo Culture of Drosophila Pupal Testis and Single Male Germ-line Cysts: Dissection, Imaging, and Pharmacological Treatment

This protocol describes the dissection and cultivation of intact testes and germ-line cysts from Drosophila melanogaster pupae. This method allows microscopic observation of spermatogenesis ex vivo. Furthermore, we describe a pharmacological assay of the effect of inhibitors on specific stages of germ-cell development in pupal testes.

해부, 이미징 및 약물 학적 치료 : 초파리의 번데기 고환 및 단일 남성 생식 줄 낭종의 생체 내 문화

During spermatogenesis in mammals and in Drosophila melanogaster, male germ cells develop in a series of essential developmental processes. This includes ...